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Abstract:

The invention relates to materials and methods for detecting interactions
between lipid complexes and lipid binding agents. More specifically, the
invention provides materials and methods for displaying lipid complexes,
particularly those containing two or more different lipid molecules, on a
hydrophobic surface so as to mimic their in vivo environment more closely
than in other analytical methods. This allows more accurate detection of
lipid complexes and even identification of lipid complexes which are not
detected by other methods. The invention lends itself particularly well
to array or microarray formats.

Claims:

1. A method comprising the steps of: (i) providing a hydrophobic support
displaying a lipid complex; (ii) contacting the lipid complex with a
sample; and (iii) detecting binding of one or more components of the
sample to the lipid complex.

2. A method according to claim 1 for detecting the presence of a lipid
binding agent in the sample.

3. A method according to claim 1 wherein a sample which is known or
suspected to contain an agent capable of binding to one or more lipid
complexes is contacted with a plurality of different lipid complexes,
said method optionally comprising detecting the presence of said agent in
said sample.

4. A method according to claim 3 comprising the step of identifying the
lipid complex or complexes to which binding occurs.

5. A method according to claim 4 wherein each of the plurality of lipid
complexes is displayed at a defined, separate location on the support.

6. A method according to claim 5 comprising identifying the location on
the support at which a positive binding reaction is obtained, and
correlating that result with the identity of the complex displayed at
that location.

7. A method according to claim 1 wherein the sample is a biological fluid
selected from the group consisting of blood, serum, plasma, cerebrospinal
fluid (CSF), saliva, mucous, or urine.

8. A method according to claim 1 wherein the sample comprises one or more
cells or viruses, an extract of a cell or virus, or a component isolated
therefrom.

9. A method according to claim 1 for use in the diagnosis of a disease
characterised by the presence of a lipid binding agent.

10. A method according to claim 9 wherein the disease is an autoimmune.

11. A method according to claim 9 wherein the disease is caused by an
infectious agent which produces a lipid binding agent.

12. A method according to claim 11 wherein the disease is cholera,
tetanus, shigellosis, botulism or influenza.

13. A method according to claim 1 for use in determining whether a test
compound in said sample is capable of binding to a lipid complex.

14. A method according to claim 13 wherein a plurality of test compounds
are tested for their ability to bind to a lipid complex of choice.

15. A method according to claim 14 wherein the hydrophobic support
carries the same lipid complex at a plurality of defined, separate
locations, and wherein each sample comprising a test compound is
contacted to an individual and distinct location on the support at which
the lipid complex is present.

16. A method according to claim 15 comprising identifying the location at
which a positive binding reaction is obtained, and correlating that
result with the identity of the test compound in the sample applied to
that location.

17. A method according to claim 16 for determining the amount of a lipid
binding agent in a sample.

18. A method according to claim 17 for use in the diagnosis of a disease
in which the levels of lipid binding agents are dysregulated.

19. A method according to claim 18 wherein the lipid binding agent is
CD82.

20. A method according to claim 1 for determining whether a lipid binding
agent binds to a particular lipid complex, wherein the sample contains a
known lipid binding agent; and wherein the method comprises the step of
detecting binding of said lipid binding agent to the lipid complex.

21. A method according to claim 20 comprising contacting the lipid
binding agent with a plurality of lipid complexes in order to determine
which complex or complexes are bound by the agent.

22. A method according to claim 21 wherein each of the plurality of
different lipid complexes is displayed at a defined, separate location on
the support.

23. A method according to claim 22 comprising identifying the location at
which a positive binding reaction is obtained, and correlating that
result with the identity of the complex displayed at that location.

24. A method of detecting the presence of a lipid complex in a sample,
the method comprising the steps of: (i) displaying the sample on a
hydrophobic support; (ii) contacting the sample with a known lipid
binding agent; and (iii) detecting binding of said lipid binding agent to
the sample.

25. A method according to claim 24 wherein the sample comprises one or
more cells, viruses, an extract of a cell or virus, or a component
isolated therefrom.

26. A method according to claim 24 wherein the sample is, or is derived
from, a tissue sample from an individual known or suspected to have a
particular disease or to be infected with a particular pathogen.

27. A method according to claim 26 wherein a single sample is tested for
reactivity with a plurality of known lipid binding agents.

28. A method according to claim 27 wherein the hydrophobic support
carries a plurality of samples at defined separate locations.

29. A method according to claim 28 wherein the hydrophobic support is
made from a material which has an advancing contact angle with respect to
water selected from the group consisting of greater than 60.degree.,
greater than 65.degree., greater than 70.degree., greater than
75.degree., greater than 80.degree., greater than 85.degree., greater
than 90.degree., greater than 95.degree., greater than 100.degree.,
greater than 105.degree., greater than 110.degree., and greater than
115.degree..

30. A method according to claim 28 wherein the hydrophobic support is
made from polyvinylidene fluoride (PVDF), polytetrafluoroethylene (PTFE),
polypropylene, polyethersulphate, polyetherimide (PEI), polyurethane,
nylon, cellulose, nitrocellulose or silica.

31. A method according to claim 24 wherein a component of the lipid
complex is selected from group consisting of a fatty acyl, a acid,
glycerolipid, glycerophospholipid, sphingolipid, sterol or prenol.

32. A method according to claim 24 wherein one or more of the lipids is
cholesterol, sphingomyelin, ceramide or digalactosyl diglyceride.

33. A method according to claim 24 wherein one or more of the lipids is a
glycolipid.

34. A method according to claim 24 wherein one or more of the lipids is a
non-glycosylated lipid.

35. A method according to claim 24 wherein the lipid complex comprises
one or more glycolipids and/or one or more glycerophospholipids.

36. (Currently amended A method according to claim 24 wherein the lipid
complex is a heterodimer or a homodimers.

37. A method according to claim 1 wherein the lipid complex contains
three, four, five, six or more lipids.

38. A method according to claim 37 wherein the lipid complex is a complex
of sulphatide, monosialoganglioside, cholesterol and
phosphatidylethanolamine.

39. A method according to claim 36 wherein the lipid complex is a
glycolipid complex and is a heterodimer of any two of the following
gangliosides GM1, GM2, GM3, GDIa, GDIb, GD3, GTIa, GTIb, GDIb, GQIb and
asialo-GM1 GM2/GT1b and GM1/GD1a.

40. A method according to claim 1 wherein the support carries every
possible homodimeric and heterodimeric combination of a set of monomeric
lipids, optionally wherein the set comprises at least 5, at least 10, or
at least 15 monomeric lipids.

41. A method according to claim 40 wherein the same lipid complex is
carried at a plurality of locations on the support.

42. A method according to claim 41 wherein the support comprises at least
100, at least 200, at least 500 or at least 1000 distinct locations each
carrying a lipid complex.

43. A method according to claim 42 wherein the support comprises at least
10, at least 20, at least 50, at least 100, at least 200, at least 300,
at least 400, or at least 500 distinct locations or lipid complexes per
square centimetre.

44. A method according to claim 43 wherein individual locations or
complexes are separated from adjacent locations or complexes by a barrier
which acts to reduce or prevent fluid flow between locations.

45. A method according to claim 44 wherein the barrier is a hydrophobic
barrier which resists fluid flow between adjacent locations or a wall.

46. A method according to claim 1 wherein the lipid binding is detected
with a molecule selected from the group consisting of an antibody, a
monoclonal antibody, serum immunoglobulin, a lectin, a siglec, a
siglec-Fc fusion protein, a bacterial toxin, a cholera toxin, a tetanus
toxin, a small molecule of 500 Da or less which possesses or is suspected
to possess the capacity to bind lipid complexes.

47. A hydrophobic support displaying a lipid complex, or a plurality of
lipid complexes at distinct defined locations.

48. A method of preparing a hydrophobic support according to claim 47,
the method comprising the step of applying the lipid complex to the
hydrophobic support.

49. A method according to claim 24 wherein the individual components of
the complex are mixed together to allow interaction before they are
applied to the support.

50. A kit for detecting binding of a lipid binding agent to a lipid
complex, the kit comprising a hydrophobic support displaying a lipid
complex according to claim 47.

51. The method as claimed in claim 10 wherein said autoimmune disease is
selected from the group consisting of Guillain Barre syndrome (GBS) and
multiple sclerosis.

Description:

FIELD OF THE INVENTION

[0001] The present invention relates to materials and methods for
detecting interactions between lipid complexes and lipid binding agents.

BACKGROUND OF THE INVENTION

[0002] Within the field of lipidomics, there is an increasing
understanding of the molecular processes by which, in the living membrane
of cells, different classes of lipid interact closely with each other and
with other membrane components, including proteins. Many classes of lipid
exist, the major ones being fatty acids, glycerolipids,
glycerophospholipids, sterol lipids including cholesterol, and
sphingolipids (Wenk, 2005). Within the category of sphingolipids, the
headgroups may be glycosylated to form classes of neutral and acidic
glycosphingolipids (GSLs). Gangliosides are a class of GSLs containing
sialic acid.

[0003] Interactions between lipids, especially GSLs, have been shown to be
functionally important in cancer cell motility and invasiveness, and in
embryogenesis (Regina and Hakomori, 2008). GSLs also act as ligands for
many bacterial toxins and for the class of cell signalling molecules
known as siglecs (sialic-acid-binding-immunoglobulin-like-lectins)
(Schiavo and van der Goot, 2001 and Crocker et al., 2007). Many
antibodies bind lipids, both in experimental situations where antibodies
have been developed as probes, and in immune and autoimmune states in
which anti-lipid antibodies can be detected in the circulation of humans
and other species. In the post-infectious inflammatory neuropathy,
Guillain Barre syndrome (GBS), anti-GSL antibodies are present. GBS is
now the leading cause of acquired paralysis. In these conditions,
antibodies which react against particular GSLs, such as gangliosides, can
sometimes be detected in patients' sera, but in many cases these cannot
be found (Willison, 2005). However, it has recently been shown that while
certain patients show reactivity to pairs of GSLs (e.g. ganglioside
complexes), antibodies from these patients fail to react with the
component species in isolation (Kaida et al., 2004).

[0004] At present, the long-established technique of enzyme-linked
immunoabsorbant assay (ELISA) is used to evaluate anti-ganglioside
antibody activity in patient sera (Willison et al., 1999), both in the
research and clinical diagnostic settings. Typically, 10 or so individual
target glycolipids are screened on 96-well polystyrene ELISA plates
against 100 microlitres of diluted patient serum per ELISA well in this
system. In some situations, accessory lipids, such as
glycerophospholipids or sterols, have be added in various ratios to
improve antibody binding to glycolipids, although this has not been
systematically evaluated to the point of being widely incorporated into
standard assay methods.

[0005] When using the ELISA technique to investigate reactivity of patient
sera to GSL complexes, a more limited panel of single GSLs applied in
pairs has been used. Even with these reduced numbers, the combinatorial
approach using 7 pairs of glycolipids required the production of over 20
different samples, applied manually and in duplicate to separate ELISA
wells, for each iteration of the experiment (Kaida et al., 2004 and Kaida
et al., 2007). If 10 or more species are used in combination, the number
of samples rapidly begins to exceed the capacity of a standard 96-well
ELISA plate and consumes so much of the patient serum test reagent that
this technique becomes impractical. When glycolipid complexes are formed
from more than 2 partners (for example, 10 glycolipids or lipids in
clusters of 4), the number of combinations rises rapidly (to 210 in this
example) and thus requires a level of miniaturisation that cannot be
achieved or practically conducted using standard 96 well ELISA plates.

[0006] There are well founded and widely recognised concerns that
interactions between lipids (e.g. glycolipids) in an artificial system,
such as ELISA, will not necessarily be representative of the interactions
that can be observed in the cell membrane in vivo (Willison, 2005). For
example, an anti-GM1 antibody may be able to detect GM1 ganglioside
immobilised in an ELISA well, but not be able to detect GM1 when it is
present in a living cell membrane. Conversely, an antibody may recognise
GM1 in a living cell membrane, but not in an ELISA. The relevance of
detection in artificial ELISA systems to in vivo biology is thus
questionable.

[0007] Single GSL dot-blot on polyvinyldifluoride (PVDF), using a manual
approach to spot individual species, has previously been described
(Chabraoui et al., 1993). Single lipids and glycolipids have also been
automatically arrayed onto PVDF membranes and probed with cerebrospinal
fluid (CSF) and serum samples from patients with multiple sclerosis
(Kanter et al., 2006). Commercially produced nitrocellulose membranes
impregnated with single glycolipids are also available (e.g.
SphingoStrips®, Molecule Probes, USA).

SUMMARY OF THE INVENTION

[0008] At its most general, the invention relates to methods for detecting
interactions between complexes of lipids and lipid binding agents.

[0009] In a first aspect, the invention provides a method comprising the
steps of: [0010] (i) providing a hydrophobic support displaying a lipid
complex; [0011] (ii) contacting the lipid complex with a sample; and
[0012] (iii) detecting binding of one or more components of the sample to
the lipid complex.

[0013] Binding of one or more components of the sample to the lipid
complex thus indicates that the sample comprises a lipid binding agent.

[0014] This method may be used to detect the presence of a lipid binding
agent in a sample. For example, it may be used to determine whether a
sample contains a binding agent capable of binding to a specific lipid
complex. A sample which is known or suspected to contain an agent capable
of binding to one or more lipid complexes may therefore be tested against
a plurality of different lipid complexes to determine whether such
binding agents are present, and to which complexes they are capable of
binding.

[0015] The method typically comprises the step of identifying the lipid
complex or complexes to which binding occurs.

[0016] The plurality of different lipid complexes may be displayed on the
same support or on a plurality of supports, depending on the format of
the assay. Where the plurality of complexes are displayed on the same
support, each complex will typically be displayed at a defined, separate
location on the support. Supports carrying a plurality of complexes in
this way may be referred to as "arrays", or "microarrays", especially
when the various locations are arranged in a regular geometric pattern.
Thus it may be possible to identify the complex or complexes to which
binding occurs by identifying the location at which a positive binding
reaction is obtained, and correlating that result with the identity of
the complex displayed at that location.

[0017] The method may be used to test a biological sample to see whether
such lipid binding agents are present. The sample may be a biological
fluid, e.g. blood (or a component thereof, such as serum or plasma),
cerebrospinal fluid (CSF), saliva, mucous, or urine. The sample may also
comprise one or more cells or other biological structures which might
contain or comprise a lipid binding agent. Cells may be animal, plant or
microbial (e.g. bacterial or fungal) cells. Other structures may include
infectious agents such as viruses. Additionally or alternatively the
sample may comprise an extract of a cell, virus, etc., or a component
isolated therefrom.

[0018] The method may thus be used for diagnosis of diseases which are
characterised by the presence of lipid binding agents. These diseases
include autoimmune diseases (e.g. Guillain Barre syndrome (GBS) and
multiple sclerosis) in which affected individuals have antibodies against
particular lipid complexes (e.g. glycolipid complexes). They may also
include diseases caused by infectious agents which produce lipid binding
agents, either on their surface or as secreted molecules, e.g. bacterial
toxins such as the cholera, tetanus, shigella and botulinum toxins, and
enzymes, such as neuraminidase, which is found on the surface of the
influenza virus. Therefore, this method may also be used for the
diagnosis of diseases such as cholera, tetanus, shigellosis, botulism and
influenza.

[0019] The methods of the invention may further be used for assessing the
repertoire of binding agents, such as natural antibodies, in normal
populations, and thereby relating this to disease susceptibility or
protective traits.

[0020] The method of the invention may also be used to determine whether a
particular test compound is capable of binding to a lipid complex. Thus
the method may be used to identify an agent capable of binding to a
specific lipid complex, for example by testing a plurality of test
compounds for their ability to bind to a lipid complex of choice. The
method may comprise selecting a test compound which is capable of binding
to the lipid complex.

[0021] This type of method may therefore be carried out by screening a
library of test compounds against the same lipid complex to see which (if
any) of the test compounds are capable of binding to it.

[0022] Thus the method may make use of a plurality of supports each
carrying the lipid complex, which may be contacted individually with
individual test compounds.

[0023] Alternatively, the method may make use of a single support carrying
the same lipid complex at a plurality of defined, separate locations. In
such cases, each test compound may be contacted to an individual and
distinct location on the support at which the lipid complex is present.
Typically it will be known which test compound was applied to each
location. Thus it may be possible to identify those test compound or
compounds capable of binding to the complex by identifying the location
at which a positive binding reaction is obtained, and correlating that
result with the identity of the test compound applied to that location.

[0024] In these embodiments of the invention the sample comprises a test
compound, which may be a small molecule (e.g. less than 500 Da in
molecular weight) or a larger molecule such as an antibody (e.g. a
monoclonal antibody), a lectin (for example a siglec or a siglec-Fc
fusion protein), or a bacterial toxin (e.g. the cholera, tetanus,
shigella or botulinum toxins). Thus, the method may be used to detect
binding of the test compound to a lipid complex. As such, this method may
be used for the identification of therapeutic or diagnostic agents, such
as antibodies, which bind to particular lipid complexes. Such agents may
be useful for the diagnosis or treatment of diseases, such as cancer, in
which lipid expression is altered (e.g. for the treatment of cancers in
which expression of glycolipids is altered, such as melanoma).

[0025] In addition, the method may also be used to determine the amount of
a lipid binding agent in a sample. Therefore, this method may be used for
the diagnosis of diseases in which the levels of lipid binding agents are
dysregulated. For example, the anti-metastasis factor CD82, which
interacts with the ganglioside complex GM2/GM3, is dysregulated in many
types of cancer.

[0026] Alternatively the method may be used to investigate the specificity
of a test compound which is already known (or suspected) to bind to lipid
complexes. Thus the invention provides a method of determining whether a
lipid binding agent binds to a particular lipid complex, the method
comprising the steps of: [0027] (i) providing a hydrophobic support
displaying the lipid complex; [0028] (ii) contacting the lipid complex
with said lipid binding agent; and [0029] (iii) detecting binding of said
lipid binding agent to the lipid complex.

[0030] The method may comprise contacting the lipid binding agent with a
plurality of lipid complexes in order to determine which complex or
complexes are bound by the agent.

[0031] The method will typically comprise the step of identifying the
lipid complex or complexes to which binding occurs.

[0032] As described above, the plurality of different lipid complexes may
be displayed on the same support or on a plurality of supports, depending
on the format of the assay. Where the plurality of complexes are
displayed on the same support, each complex will typically be displayed
at a defined, separate location on the support. It will therefore be
possible to identify the complex to which the agent binds by identifying
the location at which a positive binding reaction is obtained, and
correlating that result with the identity of the complex displayed at
that location.

[0033] This method may be used to identify lipid complexes bound by known
lipid binding agents, including lectins (e.g. siglecs) and antibodies
(e.g. serum immunoglobulins and monoclonal antibodies). In particular, if
the lipid complex identified by this method is known to be aberrantly
expressed in a particular disease, such as a cancer, the method may be
used to identify particular lipid binding agents as potential therapeutic
or diagnostic agents. Alternatively, if a particular lipid binding agent
is known to specifically recognise a particular type of cell or disease
state, this method may be used to identify the lipid complex to which it
binds, thus identifying that complex as a marker of that cell or disease
state.

[0034] According to a further aspect of the invention, there may be
provided a method of detecting the presence of a lipid complex in a
sample, the method comprising the steps of: [0035] (i) displaying the
sample on a hydrophobic support; [0036] (ii) contacting the sample with a
known lipid binding agent; and [0037] (iii) detecting binding of said
lipid binding agent to the sample.

[0038] Binding of the known lipid binding agent to the sample thus
indicates the presence of a lipid complex in the sample.

[0039] This method may be used for the identification of cells known to
carry distinctive lipid complexes not found in other cell types, or to
possess distinctive quantities of a particular lipid complex (e.g.
increased or reduced) compared to other cell types. Thus the method may
be used (for example) for diagnosis of a disease in which lipid complexes
are aberrantly expressed on the cell surface (e.g. certain types of
cancer), or for identification of a pathogen.

[0040] The sample may therefore comprise one or more biological cells or
an infectious agent such as a virus, or an extract thereof (such as a
membrane fraction, e.g. a plasma membrane fraction). Cells may be animal,
plant or microbial (e.g. bacterial or fungal) cells. For example the
sample may be, or may be derived from, a tissue sample from an individual
known or suspected to have a particular disease (e.g. cancer) or to be
infected with a particular pathogen (e.g. bacterium, virus or other
infectious agent).

[0041] It will clearly be possible to test a single, sample for reactivity
with a plurality of known lipid binding agents. This can be done using a
single support carrying a plurality of lipid complexes at defined
separate locations as already described.

[0042] To increase throughput, it may also be desirable to analyse a
number of different samples (e.g. samples from different individuals, or
even different samples from the same individual) on the same support. A
single support may therefore be used to test a plurality of samples, each
against a plurality of lipid complexes (which may be the same or
different for each sample). The skilled person will be capable of
designing a suitable format for the support, given the teaching in this
specification.

[0043] The invention also extends to materials for use in the
above-described methods, as well as methods for their production.

[0044] Thus according to a further aspect of the invention, there is
provided a hydrophobic support displaying a lipid complex. It may display
a plurality of lipid complexes at distinct defined locations, which
complexes may be same or different as described elsewhere in this
specification. This hydrophobic support may be used in the methods of the
invention described above. Individual locations or complexes may be
separated from adjacent locations or complexes by a barrier which acts to
reduce or prevent fluid flow between locations, thus preventing
cross-contamination in the course of preparing a support or in performing
an assay. The barrier may be a hydrophobic barrier of a wax or other
suitable material which resists fluid flow between adjacent locations.
Alternatively adjacent locations may be separated by a wall. In such
embodiments, each individual location may be surrounded by a wall
separating it from adjacent locations, so that each location can be
regarded as being (or being located within) an individual well on the
support. Thus the walls may be arranged in a grid pattern depending on
the configuration of the support and the locations thereon.

[0045] According to a further aspect of the invention, there may be
provided a method of displaying a lipid complex on a hydrophobic support,
the method comprising the step of applying the lipid complex to the
hydrophobic support. This step of applying the lipid complex to the
hydrophobic support may be automated. Preferably the individual
components of the complex are mixed together to allow interaction before
they are applied to the support. This further facilitates interactions
which more accurately reflect those seen in natural biological
environments.

[0046] According to a further aspect of the invention, there may be
provided a kit for detecting binding of a lipid binding agent to a lipid
complex, the kit comprising a hydrophobic support displaying a lipid
complex. The hydrophobic support may, for example, display a plurality of
lipid complexes. The kit may also include positive and negative control
reagents, detection reagents and/or methodology including software for
reading, analysing and interpreting the array, as set out in more detail
below. This kit may be used in any of the methods of the invention.

[0047] The following embodiments relate to any of the aspects of the
invention described above.

[0048] The hydrophobic support may be made from a material which has an
advancing contact angle with respect to water of greater than 60°,
greater than 65°, greater than 70°, greater than
75°, greater than 80°, greater than 85°, greater
than 90, greater than 95°, greater than 100°, greater than
105°, greater than 110°, or greater than 115°.

[0049] The hydrophobic support may be made from a material which has an
advancing contact angle with respect to water of greater than 75°,
greater than 80°, greater than 85°, or greater than
90°.

[0050] Examples of suitable materials for making the hydrophobic support
may include polyvinylidene fluoride(PVDF), polytetrafluoroethylene
(PTFE)/Teflon', polypropylene, polyethersulphate, polyetherimide (PEI),
polyurethane, nylon, cellulose, nitrocellulose or silica. For example,
the hydrophobic support may be made from a PVDF slurry, or a silica
slurry. The hydrophobic support may be a membrane, which may be formed
from any of the materials listed above. A SphingoStrip® may be
suitable.

[0051] The lipid complex comprises two or more lipids. Each component may,
for example, be a fatty acyl (e.g. fatty acid), glycerolipid,
glycerophospholipid, sphingolipid, sterol or prenol.

[0052] For example, one or more of the lipids may be cholesterol,
sphingomyelin, ceramide or digalactosyl diglyceride.

[0053] In some embodiments, as described in more detail below, one or more
of the components of the complex may be a glycolipids. The complex may
also contain one or more non-glycosylated lipids. Alternatively, the
complex may comprise only glycolipids or only non-glycosylated lipids.

[0054] For example, the lipid complex may comprise one or more glycolipids
(which have a carbohydrate component and a lipid component) and/or one or
more glycerophospholipids. Bacterial lipids and glycolipids, such as
lipopolysaccharide from Pseudomonas aeruginosa or lipooligosaccharide
from Campylobacter jejuni may also be included.

[0055] Thus the lipid complex may, for example, comprise two or more
glycolipids, a glycolipid and a non-glycosylated lipid (e.g. cholesterol,
sphingomyelin or phosphatidylcholine, or a complex of two or more lipids,
such as cholesterol and phosphatidylethanolamine), or two or more
non-glycosylated lipids, which may include sphingosine or phosphatidyl
components. As such, the lipid complexes may be heterodimers, or
homodimers. The lipid complex may include two or more, three or more,
four or more, five or more, or six or more lipids (e.g. the lipid complex
may be a complex of sulphatide, monosialoganglioside, cholesterol and
phosphatidylethanolamine). The method may include the step of mixing
together two or more of the individual components of the complex, and
preferably all of the individual components of the complex, before they
are applied to the hydrophobic support. In some embodiments, the
glycolipid complex may be displayed on the hydrophobic support in
duplicate.

[0056] As discussed above, each lipid in the lipid complex may be a
glycolipid, for example, a glycosphingolipid, such as a ganglioside. The
glycolipid complexes may be heterodimers. For example, they may be
heterodimers of any two of the following gangliosides: GM1, GM2, GM3,
GD1a, GD1b, GD3, GT1a, GT1b, GD1b, GQ1b and asialo-GM1 (e.g. GM2/GT1b and
GM1/GD1a).

[0057] The lipid complex may be displayed on the hydrophobic support at a
distinct defined location. Furthermore, a plurality of different
glycolipid complexes may be displayed on the hydrophobic support, each at
a distinct defined location on the hydrophobic support.

[0058] For example, the support may carry every possible homodimeric and
heterodimeric combination of a given set of monomeric lipids. The set may
comprise at least 5, at least 10, at least 15, or more monomeric lipids.
Each combination may be displayed at one, two, three or even more
locations.

[0059] The support may carry one or more replicates of a chosen set of
lipid complexes.

[0060] Additionally or alternatively, a plurality of locations on the
support may each carry the same lipid complex, in order to facilitate
screening of a plurality of test compounds (e.g. a library of compounds)
for binding activity towards a chosen complex.

[0061] On a single support, there may be at least 100, at least 200, at
least 500, at least 1000, or even more distinct locations each carrying a
lipid complex. A particular advantage of the supports described herein is
that the assay format can be significantly miniaturised as compared, for
example, to a conventional ELISA format. Thus the number of complexes (or
locations) per unit area of the support may be significantly larger than
is possible in the ELISA format. For example, a single support may have
at least 10, at least 20, at least 50, at least 100, at least 200, at
least 300, at least 400, at least 500, or even more distinct locations or
complexes per square centimetre. These locations or complexes may be in a
grid format and such grids may include, for example, more than 5×5,
more than 10×10, more than 20×20, more than 30×30, more
than 40×40, or more than 50×50 locations or complexes per
square centimetre. Each distinct location may therefore have an area of
less than 1.0 mm2, less than 0.5 mm2, less than 0.2 mm2,
less than 0.1 mm2, less than 0.05 mm2, less than 0.02 mm2,
or less than 0.01 mm2. If the lipid complexes are spotted on to the
support as substantially circular spots, each spot may, for example, have
a diameter of less than 1.0 mm, less than 0.5 mm, less than 0.2 mm, less
than 0.1 mm, less than 0.05 mm, less than 0.02 mm, or less than 0.01 mm.

[0062] The skilled reader will understand that combinations and variations
of these various arrangements are also possible.

[0063] Knowing the location of each complex on the support, and/or the
location at which any given test compound is applied, permits
identification of a lipid complex bound by any particular lipid binding
agent, or the test compound capable of binding to any given lipid
complex.

[0064] The lipid binding agent may be, for example, an antibody (e.g. a
monoclonal antibody or serum immunoglobulin), a lectin, which may be
mammalian, bacterial or plant lectin, (such as a siglec or siglec-Fc
fusion protein), a bacterial toxin (e.g. the cholera or tetanus toxin),
or any other suitable protein. Other types of molecule such as
carbohydrates or nucleic acids (e.g. aptamers) may also be used, as may
small molecules (e.g. of 500 Da or less) which possess or are suspected
to possess the capacity to bind lipid complexes. As will already be
apparent, the methods of the invention may be used to screen a library of
any suitable type of compound for lipid binding ability.

[0065] The invention will now be described in detail, by way of example,
with reference to the accompanying figures.

[0067] FIG. 2 shows examples of processed membranes. FIG. 2A shows a
10×10 GSL combinatorial grid that has been probed with a serum from
a patient with an inflammatory neuropathy. FIGS. 2B and 2C show
23×23 combinatorial lipid grids. The lipid names have been replaced
by numbers, as shown in FIG. 1B, using the grid key shown in FIG. 1C. In
FIG. 2B, the grid has been probed with serum from a patient with an
undiagnosed neurological disorder. In FIG. 2C, the grid has been probed
with serum from a patient with multiple sclerosis.

[0068] FIG. 3 shows processed membrane arrays developed on X-ray film.
These arrays show ganglioside series GSLs illustrating three alternative
patterns of binding. In FIG. 3A, siglec-E was used as the probe; in FIG.
3B, the monoclonal antibody mAb MOG26 was used as the probe; and in FIG.
3C, cholera toxin was used as the probe.

[0070] FIG. 5 shows the differing responses of anti-GM1 mAbs DG1 and DG2
to complexes of gangliosides containing GM1. (A) Illustrative ELISA
plates. The ganglioside complex absorbed to each well is established by
combining the row and column headings. Wells labelled with `x` are
negative controls (methanol only). (B) Illustrative PVDF glycoarrays. DG1
was used as the primary antibody for the left hand membrane, DG2 on the
right. The circles enclose duplicate spots of GM1 alone, hexagons denote
GM1/GD1a complex. (C) Quantitative ELISA results from 4 independent
experiments. (D) Quantitative results from the PVDF glycoarray (n=3). (E)
Comparison of the inhibitory effect of GD1a on GM1 binding for DG1 and
DG2. *p=0.02 for two sided T-test of DG1 v DG2. For all graphs DG1 is
represented by filled bars, DG2 by open bars.

[0075] Various of these types of lipid possess head groups which comprise
one or more carbohydrate moieties. Such lipids will be referred to herein
as glycolipids, whichever category of lipid mentioned above they may
belong to. Similarly, lipid types which do not contain a carbohydrate
moiety will be referred to as non-glycosylated lipids.

[0076] Glycolipids may be of particular interest, and comprise a lipid
component and a carbohydrate component. Typically, the carbohydrate
component forms a polar head-group and the lipid component forms a lipid
tail. In nature, glycolipids occur in diverse membrane environments in
most species. In the cell membranes of eukaryotes, the carbohydrate
element is associated with phospholipids on the exoplasmic surface of the
cell membrane and extends from the phospholipid bilayer into the aqueous
environment outside the cell.

[0077] Examples of glycolipids include galactolipids, and
glycosphingolipids (GSLs), such as cerebrosides, gangliosides,
globosides, sulphatides, and glycophosphospingolipids. Glycolipids are
generally synthesised from ceramides and sphingosine bases. Bacteria and
other organisms also produce a number of well known glycolipids, such as
lipopolysaccharide from Pseudomonas aeruginosa and lipooligosaccharide
from Campylobacter jejuni.

[0078] Gangliosides are the most complex mammalian glycolipids and contain
negatively charged oligosaccharides with one or more sialic acid
residues. They are highly expressed in nerve cells, but are also present
in plasma membrane in all other sites throughout the mammalian system.
Specific examples of gangliosides include GM1, GM2, GM3, GD1a, GD1b, GD3,
GT1a, GT1b, GD1b, GQ1b and GQ1alpha. Asialo-GM1 is a similar structure to
GM1, but does not contain sialic acid.

Lipid Complexes

[0079] A lipid complex comprises two or more lipids physically associated
with one another. Thus complexes may comprise one or more of any of the
various categories of lipid described above, including glycolipids, fatty
acids, glycerolipids, glycerophospholipids, and sterols.

[0080] A lipid complex may include two or more, three of more, four or
more, five or more, or six or more lipids. Each of the individual
components may be from any of the categories described. (For example, a
single complex may be a complex of sulphatide, monosialoganglioside,
cholesterol and phosphatidylethanolamine.)

[0081] In certain embodiments, the complex may comprise at least one
glycolipid. Other components of the complex may be non-glycosylated
lipids or glycolipids.

[0082] Thus a lipid complex may, for example, comprise two or more
non-glycosylated lipids (which may include sphingosine or phosphatidyl
components), two or more glycolipids (such as gangliosphingolipids, e.g.
gangliosides), or it may comprise a glycolipid and a non-glycosylated
lipid (e.g. cholesterol, sphingomyelin or phosphatidylcholine.

[0083] When two or more lipids form a complex, this complex can contain
binding sites for lipid binding agents which cannot be recognised or
bound by binding agents applied to the monomers. Similarly, when two or
more different lipids (whether or not of the same category) form a
complex, this complex can contain binding sites for lipid binding agents
which cannot be recognised or bound by binding agents applied to
homogeneous complexes of the individual components. Therefore, it is
believed that heterologous complexes (i.e. heterodimeric complexes and
higher order complexes of two or more different lipids), may possess
binding sites for lipid binding agents which are not found on the
monomers themselves, or on homogeneous (e.g. homodimeric) complexes of a
single lipid. However, the methods of the invention may nevertheless find
use with homodimer lipid complexes.

[0084] A glycolipid complex may comprise two or more, three or more, four
or more, five or more, or six or more glycolipids, such as
glycosphingolipids, e.g. gangliosides. However, the glycolipid complex is
not limited to a mixture of the same type of molecule. For example, the
glycolipid complex may be a mixture of a glycosphingolipid and a
ganglioside. These glycolipid complexes may be heterodimers. For example,
they may be heterodimers of any two of the following gangliosides: GM1,
GM2, GM3, GD1a, GD1b, GD3, GT1a, GT1b, GD1b, GQ1b and asialo-GM1 (e.g.
GM2/GT1b and GM1/GD1a).

[0085] The individual components of the lipid complex may be mixed before
they are used in the methods described herein. This allows interaction of
the lipids before they are applied to the hydrophobic support. This
facilitates interactions between the lipids which more accurately reflect
those seen in vivo.

Lipid Binding Agents

[0086] Lipid binding agents are agents which bind to lipids. Typically,
they bind at least in part to a head group on the lipid. This headgroup
may include a wide range of chemical modifications, such as inositol,
glycerol and phosphate groups. On glycolipids, this head group is or
comprises a carbohydrate molecule.

[0087] A range of agents may act as lipid binding agents. Examples of such
agents which occur naturally include antibodies, lectins (e.g. siglecs)
and bacterial toxins (e.g. the cholera, tetanus, shigella or botulinum
toxins). The methods of the invention extend to use of, and screening
for, agents which do not occur in vivo, such as small molecules capable
of binding to particular complexes, monoclonal antibodies, nucleic acids
(e.g. aptamers), etc. As already described, the methods of the invention
may be used to screen a library of any suitable type of compound for
lipid binding ability.

Samples

[0088] As described herein, the methods of the invention may be used to
detect the presence of a lipid binding agent in a sample, or to detect
the presence of a given lipid complex in a sample. Suitable samples for
use in such methods include biological fluids and tissue samples taken
from individuals affected by, or suspected of being affected by,
particular conditions as well as samples containing, or isolated from
other types of organism or infectious agent such as microbial (bacterial
or fungal) cells and viruses. Suitable biological fluids include blood
(and components thereof including serum and plasma), urine, saliva,
mucous and cerebrospinal fluid (CSF). These individuals may be healthy
individuals, or may be suspected of having an autoimmune disease, such as
Guillain Barre syndrome (GBS), multiple sclerosis, or an infectious
disease, such as cholera or influenza.

[0090] In some embodiments, the sample for use in this detection method
may include a test compound, such as an antibody (e.g. a monoclonal
antibody), a siglec (e.g. a siglec-Fc fusion protein), or a bacterial
toxin (e.g. the cholera or tetanus toxin).

Hydrophobic Supports

[0091] Hydrophobic supports displaying lipid complexes are used in the
methods of the present invention. The hydrophobic support may be made
from a material which has an advancing contact angle with respect to
water of greater than 60°, greater than 65°, greater than
70°, greater than 75°, greater than 80°, greater
than 85°, greater than 90°, greater than 95°,
greater than 100°, greater than 105°, greater than 1100, or
greater than 115°.

[0092] The hydrophobic support may be made from a material which has an
advancing contact angle of greater with respect to water than 75°,
greater than 80°, greater than 85°, or greater than
90°.

[0093] The advancing contact angle of a material with respect to water can
be measured, for example, by depositing a water drop (e.g. having a
volume of about 2 μl) on the surface of the material using, for
example, a syringe. The advancing contact angle at the interface between
the water drop and the material can then be measured using a contact
angle meter, such as a contact angle goniometer.

[0094] Examples of suitable materials may include polyvinylidene
fluoride(PVDF), polytetrafluoroethylene (PTFE)/Teflon®, polypropylene,
polyethersulphate, polyetherimide (PEI), polyurethane, nylon, cellulose,
nitrocellulose or silica. For example, the hydrophobic support may be
made from a PVDF slurry, or a silica slurry. The hydrophobic support may
be a membrane, which may be formed from any of the materials listed
above. A SphingoStrip® may be suitable.

[0095] The hydrophobic support may itself be supported on a solid
substrate, which may be made from any suitable material such as plastics
material or glass. In a particularly convenient format, the hydrophobic
support is supported on a conventional glass microscope slide (e.g. 6
cm×2 cm). Thus the support may be formed by applying a PVDF slurry
or a silica slurry to a glass microscope slide and allowing the slurry to
solidify into a membrane.

[0096] Without wishing to be bound by any particular theory, it is
believed that the hydrophobic lipid components of the lipids tend to
associate with, or interact with, the hydrophobic support via hydrophobic
interactions (van der Waals interactions). Therefore, binding of lipid
complexes to the hydrophobic supports is facilitated by the hydrophobic
nature of these supports. Some classes of lipids comprise a polar head
group, such as a inositol, glycerol, phosphate or carbohydrate group, in
addition to their lipid component. For example, glycolipids comprise a
hydrophobic lipid component and a carbohydrate component, which is
typically polar and/or charged. Therefore, any polar head group (e.g. a
carbohydrate component, such as an oligosaccharide chain) is typically
not anchored to the hydrophobic support, and is (to some extent at least)
free to move and interact with neighbouring head groups. Interaction
between neighbouring head groups (e.g. oligosaccharide chains) is thought
to be important for the formation of lipid complexes in a manner that may
be representative of the situation found in biological membranes. In
particular, this orientation of the lipids (e.g. glycolipids) may also
improve accessibility of their head groups (e.g. carbohydrate components)
for binding to lipid binding agents, which may assist in the binding
required for the methods of the invention. In particular, glycolipid
binding agents usually bind to the carbohydrate components of glycolipid
complexes, although the binding site may in some cases extend onto the
lipid component of the complex.

[0097] The presence of a non-glycosylated lipid in a glycolipid complex
(to form a non-glycosylated lipid lipid/glycolipid complex) may further
aid the stabilisation of the complex in a manner that permits binding of
a binding agent that may not be evident using other methods. Complexes
made up entirely of non-glycoslyated lipids (e.g. non-glycosylated lipid
dimers) may also be displayed on hydrophobic supports and may this may
permit binding of lipid-binding agents that may not be evident using
other methods.

[0098] Therefore, the orientation of lipid complexes (e.g. glycolipid
complexes) bound to hydrophobic supports, as well as the interactions
between each lipid in the complex, is thought to be more representative
of the in vivo situation than when other test systems (such as ELISA) are
used.

[0099] The application of lipid complexes to the hydrophobic support may
be automated. Each lipid complex is preferably applied to the hydrophobic
support in duplicate (e.g. as duplicate spots). The lipid complexes may
be spotted onto the hydrophobic support, preferably at a distinct,
defined location. This allows identification of a lipid complex bound by
a lipid binding agent through correlation of a positive binding reaction
with a particular location on the hydrophobic support. Preferably two or
more of the individual lipid components (and preferably all of the
components) are mixed together and allowed to interact with one another
before they are applied to the support.

[0100] The lipid complexes may be applied to the hydrophobic support in a
particular pattern or configuration. For example, a plurality of lipid
complexes may be applied to the hydrophobic support in a regular array
(e.g. a grid).

[0101] Suitable grid matrices include, for example, 10×10
(rows×columns), 20×20, 23×23, 30×30, 40×40,
50×50, 100×100 and 200×200 grids. Preferably, the grid
matrices are up to 1000×1000, up to 500×500, up to
200×200, up to 100×100, or up to 50×50 in size. The
hydrophobic support may, for example, be supported on a solid substrate,
such as glass. For example it may be attached to a conventional glass
microscope slide (e.g. 20×60 mm). Grids can, however, be much
larger than this (e.g. 200×200 mm) for more complex applications,
especially when complexes comprising more than two glycolipids are used.

[0102] A single support may comprise at least 100, at least 200, at least
500, at least 1000 or even more distinct locations, each carrying a lipid
complex. A particular advantage of the assay format of the invention is
that it can be significantly miniaturised as compared, e.g. to a
conventional ELISA format. Thus a much higher density of complexes may be
applied and tested per unit area of the support. For example, a single
support may have at least 10, at least 20, at least 50, at least 100, at
least 200, at least 300, at least 400, at least 500 or even more distinct
locations or complexes per square centimetre. These locations or
complexes may be in a grid format and such grids may include, for
example, more than 5×5, more than 10×10, more than
20×20, more than 30×30, more than 40×40, or more than
50×50 locations or complexes per square centimetre. Each distinct
location may therefore, have an area of less than 1.0 mm2, less than
0.5 mm2, less than 0.2 mm2, less than 0.1 mm2, less than
0.05 mm2, less than 0.02 mm2, or less than 0.01 mm2. If
the lipid complexes are spotted on to the support as substantially
circular spots, each spot may, for example, have a diameter of less than
1.0 mm, less than 0.5 mm, less than 0.2 mm, less than 0.1 mm, less than
0.05 mm, less than 0.02 mm, or less than 0.01 mm.

Detection Methods

[0103] Disclosed herein is a method comprising the steps of: (i) providing
a hydrophobic support displaying a lipid complex; (ii) contacting the
lipid complex with a sample; and (iii) detecting binding of one or more
components of the sample to the lipid complex.

[0104] Binding of one or more components of the sample to the lipid
complex thus indicates that the sample comprises a lipid binding agent.

[0105] The method may also include one or more of the following steps.

[0106] (a) The method may include the step of mixing two or more
individual components of the lipid complex (and preferably all of the
components of the complex) before they are applied to the hydrophobic
support. This allows interaction between the components before they are
applied to the hydrophobic support, thus facilitating interactions which
more accurately reflect those seen in vivo.

[0107] (b) The method may include the step of applying a blocking solution
containing a component which does not have substantial lipid binding
ability (e.g. a protein, such as bovine serum albumin) to the hydrophobic
support before contacting the support with the sample. This can help to
prevent non-specific interaction between lipid binding substances or
other substances in the sample and the support and/or lipid complexes
displayed thereon.

[0108] (c) The method may include the step of applying the lipid complex
to the hydrophobic support, or the method may make used of a hydrophobic
support onto which a lipid complex has already been applied. The
hydrophobic support displaying the lipid complex is then contacted with a
sample to be screened for the presence of lipid binding agents.

[0109] (d) The method may also include the step of identifying the
location of the lipid complex bound by a lipid binding agent and
correlating this location with the identity of the lipid complex. This
permits identification of the lipid complex bound by the lipid binding
agent, especially if the lipid complex is displayed on the hydrophobic
support at a defined distinct location.

[0110] Various methods can be used to detect such binding. For example, a
labelled antibody (e.g. an antibody linked to horse radish peroxidise
(HRP)) can be used to detect agents bound to the lipid complexes. Binding
of this labelled antibody can then be detected using, for example, a
chemiluminescence reaction. Alternatively, components of the sample (e.g.
siglec-Fc fusion proteins or bacterial toxin conjugates) can be directly
labelled, e.g. by conjugation to HRP, or to an HRP-liked anti-Fc
antibody, in the case of siglec-Fc fusion proteins. Binding of these
agents to the lipid complexes displayed on the hydrophobic support can
then be detected using, for example, a chemiluminescent reaction.
Alternatively, binding can be detected using chemifluorescence reactions.
Chemifluorescence can, for example, be detected using a phosphoimager. In
addition, fluorescently labelled secondary antibodies may be used to
detect lipid binding agents bound to the lipid complexes displayed on the
hydrophobic support.

[0111] This detection method may be used to detect the presence of lipid
binding agents (such as antibodies) in a sample taken from a patient
(e.g. a serum sample or a sample of CSF). Therefore, this method can be
used for the diagnosis of a variety of autoimmune diseases, such as
Guillain-Barre syndrome (GBS), in which auto-antibodies against
particular glycosphingolipids (typically gangliosides) are produced, or
multiple sclerosis.

[0112] This detection method can also be used for the detection of
diseases caused by infectious agents which produce lipid binding agents,
either on their surface or as secreted molecules, e.g. bacterial toxins
such as the cholera, tetanus, shigella and botulinum toxins and enzymes,
such as neuraminidase (which is found on the surface of the influenza
virus). Therefore, this method may also be used for the diagnosis of
diseases, such as cholera, tetanus, shigellosis, botulism and influenza,
which are caused by infectious agents.

[0113] For example, cholera toxin binds to GM1 series complexes and
tetanus toxin binds to ganglioside complexes including GD3/GM2 and
GD3/GD1a, as well as GD1b and GT1b series complexes. Therefore, a method
for the detection or diagnosis of cholera may involve the use of a
hydrophobic support which displays one or more GM1 ganglioside complexes.
Similarly, a kit for the detection or diagnosis of cholera may comprise a
hydrophobic support which displays one or more GM1 ganglioside complexes.
A method for the detection or diagnosis of tetanus may involve the use of
a hydrophobic support which displays one or more GD3/GM2, GD3/GD1a, GD1b
and/or GT1b ganglioside complexes. Similarly, a kit for the detection or
diagnosis of tetanus may comprise a hydrophobic support which displays
one or more GD3/GM2, GD3/GD1a, GD1b and/or GT1b ganglioside complexes.

[0114] These diagnostic methods can include the step of obtaining a sample
from a patient (such as blood sample (e.g. a serum sample), or a urine,
saliva, mucous or CSF sample), or can be carried out using a sample that
has already been obtained from a patient. This sample is then used to
contact a hydrophobic support comprising displaying a lipid complex.

[0115] Binding of one or more antibodies in the sample to be screened to
one or more of the lipid complexes displayed on the hydrophobic support
may indicate that the patient has a disease, such as an autoimmune
disease, e.g. GBS or multiple sclerosis. The presence of a lipid binding
agent in the sample may also indicate the susceptibility of an individual
to developing a disease or provide an indication of the level of immunity
present against an infectious agent.

[0116] Such binding of antibodies to the lipid complexes arrayed on the
hydrophobic support can be detected using various methods, as described
in the "Methods of screening" section above. For example, a labelled
antibody (e.g. an antibody linked to horse radish peroxidise (HRP)) can
be used to detect antibodies bound to the lipid complexes. Binding of
antibodies to the lipid complexes arrayed on the hydrophobic support can
then be detected, for example using a chemiluminescent reaction.
Alternatively, binding can be detected using chemifluorescence reactions
using, for example, a phosphoimager. In addition, fluorescently labelled
secondary antibodies may be used to detect antibodies bound to the lipid
complexes arrayed on the hydrophobic support.

[0117] This detection method may also be used to detect binding of a test
compound in a sample to a lipid complex. The test compound may be an
antibody (e.g. a monoclonal antibody), lectin (such as a siglec, e.g. a
siglec-Fc fusion protein), a bacterial toxin (e.g. the tetanus or cholera
toxins), or anylectin, protein or nucleic acid. Thus, the method may be
used to detect binding of the test compound to a lipid complex. As such,
this method may be used for the identification of therapeutic agents,
such as therapeutic antibodies, which bind to particular lipid complexes.
Therapeutic agents identified by this method may be useful for the
treatment of diseases, such as cancer, in which lipid expression is
altered (e.g. for the treatment of cancers in which expression of
glycolipids is altered). For example, melanoma cells display altered
ganglioside profiles (Lloyd et al., 1982).

[0118] In addition, the method may also be used to determine the amount of
a particular lipid binding agent in a sample. Therefore, this method may
be used for the diagnosis of diseases in which the levels of lipid
binding agents are dysregulated. For example, the anti-metastasis factor
CD82, which interacts with the ganglioside complex GM2/GM3, is
dysregulated in many cancers (Regina and Hakamori, 2008).

[0119] The invention also relates to methods of determining whether a
known lipid binding agent binds to a particular lipid complex, the method
comprising the steps of: (i) providing a hydrophobic support displaying
the lipid complex; (ii) contacting the lipid complex with said lipid
binding agent; and (iii) detecting binding of said lipid binding agent to
the lipid complex.

[0120] Binding of said lipid binding agent to the lipid complex thus
indicates that the known lipid binding agent binds the lipid complex.

[0121] This method may be used to identify lipid complexes bound by known
lipid binding agents (such as siglecs (e.g. lectins) and monoclonal
antibodies). In particular, if the lipid complex identified by this
method is known to be aberrantly expressed in a particular disease, such
as cancer (e.g. a melanoma), the method may be used to identify known
lipid binding agents as potential therapeutic agents.

[0122] As described above, this method may include the step of mixing the
individual components of the lipid complex before they are applied to the
hydrophobic support and/or the step of applying a blocking solution to
the hydrophobic support before contacting the support with the sample.

[0123] This method may also include the step of applying the lipid complex
to the hydrophobic support, or the method may make use of a hydrophobic
support onto which a lipid complex has already been applied. The
hydrophobic support displaying the lipid complex is then contacted with a
sample to be screened for the presence of lipid binding agents.

[0124] The method may also include the step of identifying the location of
the lipid complex bound by a lipid binding agent and correlating this
location with the identity of the lipid complex. This permits
identification of the lipid complex bound by the lipid binding agent,
especially if the lipid complex is displayed on the hydrophobic support
at a defined distinct location.

[0125] As described above, various methods can be used to detect such
binding of the known lipid binding agent to the lipid complex. For
example, a labelled antibody (e.g. an antibody linked to horse radish
peroxidise (HRP)) can be used to detect agents bound to the glycolipid
complexes. Binding of this labelled antibody can then be detected using,
for example, a chemiluminescence reaction. Alternatively, components of
the sample (e.g. siglec-Fc fusion proteins or bacterial toxin conjugates)
can be directly labelled, e.g. by conjugation to HRP, or to an HRP-liked
anti-Fc antibody, in the case of siglec-Fc fusion proteins. Binding of
these agents to the lipid complexes displayed on the hydrophobic support
can then be detected using, for example, a chemiluminescent reaction.
Alternatively, binding can be detected using chemifluorescence reactions.
Chemifluorescence can, for example, be detected using a phosphoimager. In
addition, fluorescently labelled secondary antibodies may be used to
detect lipid binding agents bound to the lipid complexes displayed on the
hydrophobic support.

[0126] The invention also relates to a method for detecting the presence
of a lipid complex in a sample, the method comprising the steps of: (i)
displaying the sample on a hydrophobic support; (ii) contacting the
sample with a known lipid binding agent; and (iii) detecting binding of
said lipid binding agent to the sample.

[0127] Binding of the known lipid binding agent to the sample thus
indicates the presence of a lipid complex in a sample.

[0128] This method may be used for the diagnosis of a disease in which
lipids are aberrantly expressed on the cell surface (e.g. certain types
of cancer, such as melanoma). For example, the sample may be a tissue
sample from a patient, or a biopsy sample.

[0129] This method may include the step of obtaining a sample from a
patient (e.g. a tissue sample, or a biopsy sample), or can be carried out
using a sample that has already been obtained from a patient. This sample
is then applied to a hydrophobic support, so that the any lipid complexes
present in the sample are displayed on the support.

[0130] As described above, various methods can be used to detect such
binding of the known lipid binding agent to the lipid complex. For
example, a labelled antibody (e.g. an antibody linked to horse radish
peroxidise (HRP)) can be used to detect agents bound to the lipid
complexes. Binding of this labelled antibody can then be detected using,
for example, a chemiluminescence reaction. Alternatively, components of
the sample (e.g. siglec-Fc fusion proteins or bacterial toxin conjugates)
can be directly labelled, e.g. by conjugation to HRP, or to an HRP-liked
anti-Fc antibody, in the case of siglec-Fc fusion proteins. Binding of
these agents to the lipid complexes displayed on the hydrophobic support
can then be detected using, for example, a chemiluminescent reaction.
Alternatively, binding can be detected using chemifluorescence reactions.
Chemifluorescence can, for example, be detected using a phosphoimager. In
addition, fluorescently labelled secondary antibodies may be used to
detect lipid binding agents bound to the lipid complexes displayed on the
hydrophobic support.

Autoimmune Diseases

[0131] Several autoimmune diseases are caused, at least partially, by the
production of auto-antibodies against particular lipids. For example,
antibodies in sera from patients with the post-infectious inflammatory
neuropathy, Guillain Barre syndrome (GBS), react against particular
glycosphingolipids (typically gangliosides). Other autoimmune diseases in
which antibodies against lipids, such as glycosphingolipids, are
produced, include multiple sclerosis. In addition, antibodies against
phospholipids, such as cardiolipin, are present in the circulation of
patients with inflammatory vascular diseases and antibodies binding to
oxidised phosphorylcholine (PC)-containing phospholipids are involved in
immune defense against microbial infections and may also be involved in
binding to self lipid components and contributing to atherosclerosis.
Therefore, such diseases can be diagnosed using the methods of the
invention.

Kits

[0132] The present invention also relates to kits which comprise a
hydrophobic support displaying a lipid complex. For example, the
hydrophobic support may be pre-printed with a plurality of lipid
complexes. The lipids are preferably glycolipids, such as
glycosphingolipids (e.g. gangliosides).

[0133] The hydrophobic support may be made from a material which has an
advancing contact angle with respect to water of greater than 60°,
greater than 65°, greater than 70°, greater than
75°, greater than 80°, greater than 85°, greater
than 90°, greater than 95°, greater than 100°,
greater than 105°, greater than 110°, or greater than
115°.

[0134] The hydrophobic support may be made from a material which has an
advancing contact angle with respect to water of greater than 75°,
greater than 80°, greater than 85°, or greater than
90°.

[0135] Examples of suitable materials may include polyvinylidene
fluoride(PVDF), polytetrafluoroethylene (PTFE)/Teflon', polypropylene,
polyethersulphate, polyetherimide (PEI), polyurethane, nylon, cellulose,
nitrocellulose or silica. For example, the hydrophobic support may be
made from a PVDF slurry, or a silica slurry. The hydrophobic support may
be a membrane, which may be formed from any of the materials listed
above. A SphingoStrip® may be suitable.

[0136] Alternatively the kit may contain the components required for a
user to synthesise their own custom array of lipid complexes and to carry
out a method of the invention. For example, the kit may comprise a
support and a panel of individual lipids (e.g. 5 or more, 10 or more, or
20 or more individual lipids). The user may then combine the panel of
lipids with one another, or with other lipids of their choice, and apply
them to the support to create their own array of lipid complexes for use
in a method of the invention. Individual locations on the support for
application of lipid complexes may be pre-printed or otherwise
pre-defined.

[0137] These kits can be used in the detection methods and diagnostic
methods described above. For example, they may be used for the diagnosis
of autoimmune diseases, such as GBS and multiple sclerosis, or infectious
diseases.

[0138] The kits may also include a positive control, i.e. a lipid binding
agent which is known to bind to at least one of the complexes present on
the membrane. For example, it may be an antibody which binds to a
glycolipid complex. The kit may also include a negative control, i.e. a
substance which is known not to bind to any of the complexes on the
membrane. Where the user prepares their own array, alternative forms of
negative control may be provided for application to the support, which
will not provide a positive result for any lipid binding agent (e.g.
methanol). The kits may also include a labelled secondary antibody (such
as an HRP-linked antibody) and/or a detection reagent to allow binding of
agents to the lipid complexes to be detected, e.g. by chemiluminescence.

Advantages Associated with Using Hydrophobic Supports to Display Lipids

[0139] There are several advantages associated with using hydrophobic
supports (such as PVDF membranes) to display lipids (e.g. glycolipids)
for detecting lipid binding proteins, rather than known techniques, such
as thin layer chromatography or ELISA.

[0140] Firstly, an automated sampler can be used to allow multiple
different combinations of lipids, e.g. glycolipids, to be spotted on to
the hydrophobic support in a highly efficient and stereotyped manner. In
contrast, preparing a large number of complexes on ELISA plates is much
slower, as well as being technically arduous. In view of the long time
taken to prepare ELISA plates with large numbers of complexes, use of
ELISA-based methods is liable to generate variation. Therefore, applying
lipid complexes to hydrophobic supports makes high throughput screening
possible.

[0141] Furthermore, as printed hydrophobic supports (e.g. PVDF membranes)
may be small (typically about 20×25 mm), only small volumes of test
solution (e.g. 250 μl) are required for each 10×10 grid. Using
ELISA, 10 ml of solution would be required for testing against the same
range of complexes (when using 100 μl/well), which represents a
forty-fold reduction in the amount of test solution required. This
miniaturisation of the method means that only a small volume of sample,
e.g. serum or other biological fluid, needs to be tested for reactivity
with a plurality of known lipid binding agents. This is important when
testing samples which have a limited availability (e.g. serum samples
from patients).

[0142] Lipids (e.g. glycolipids) bind to hydrophobic supports (e.g. PVDF
membranes) via a hydrophobic interaction with their lipid components
(e.g. lipid tail). Therefore, lipids are displayed on hydrophobic
supports (e.g. PVDF membranes) in a way that may be more similar to their
in vivo orientation. As the head group component (e.g. the carbohydrate
component in glycolipids) is not anchored, interaction with neighbouring
head group components, which is crucial to the formation of lipid (e.g.
glycolipid) complexes, is permitted. In ELISA, depending on the type and
composition of the microtitre plate used, lipids (e.g. glycolipids) may
bind to the ELISA plate through electrostatic interactions with their
head-groups. Therefore, lipid may be displayed differently on ELISA
plates in comparison with PVDF or other hydrophobic membranes.

[0143] As a consequence of the orientation of lipids (e.g. glycolipids)
when displayed (e.g. as an array) on a hydrophobic support, such as a
PVDF membrane, the combinatorial glycoarray technique of the present
invention has the potential to reflect a different pattern of
carbohydrate-carbohydrate and other head group interactions in comparison
with ELISA techniques. This pattern of binding to PVDF may in some
circumstances better reflect the topographical organisation of lipids
(e.g. glycolipids) occurring in living hydrophobic supports (e.g. cell
membranes) more accurately than known techniques, such as ELISA. In
support of this, experimental data described herein confirm that
reactivities of certain glycolipid binding antibodies seen on PVDF are
not always consistent with those detected on ELISA, particularly with
respect to anti-complex activity.

[0144] For example, as shown in FIG. 5, a monoclonal antibody (DG1)
previously generated by the present inventors reacts significantly with a
mixture of GM1/GD1a on ELISA, but not at all with the same complex on
PVDF. It also fails to bind at all to living tissue in which GM1/GSL
complexes are thought to form (see FIG. 6). In contrast, the antibody DG2
binds GM1 in complex with GD1a and in complex with other GSLs in both
ELISA and PVDF glycoarrays (showing that both methods are functioning
well in this controlled experiment). DG2 is also able to bind GM1 in
living tissue, as shown in FIG. 6. Thus, the PVDF array is able to
identify an antibody (DG1), which does not bind GM1 in tissue, as being
unable to bind to GM1 complexes, whereas the ELISA is not able to
discriminate this as effectively.

[0145] Another example demonstrating the inconsistency of results obtained
using ELISA and PVDF is shown in FIG. 7. In this example, the monoclonal
antibody MOG26 binds a GM1/GD1a complex on ELISA, but not on PVDF.
Consistent with the results obtained using PVDF, MOG26 binds live tissue
in transgenic mice which express complexes of GM1/GD1a (data not shown).

[0146] These examples strongly suggest that using PVDF-based methods to
display lipid complexes (e.g. glycolipid complexes) is likely to be more
representative of the in vivo situation than using ELISA-based assays.

[0148] Stock solutions of each of the above were prepared in a 50:50 (v/v)
chloroform:methanol mixture, at 1 to 10 mg/ml. Working solutions were
made by further dilution in methanol to 0.1 mg/ml. For single samples,
200 μl of the working solution was added to a 300 μl capacity
micro-sampling vial (Chromacol, UK). To create complexes, 100 μl of
each constituent GSL was added to a vial. Vials were sealed using caps
with a rubber insert (Chromacol, UK), allowing puncture by the
autosampler needle. All samples were then sonicated for 3 minutes prior
to use.

[0149] Sheets of PVDF membrane (Sigma, UK) were cut into 20×25 mm
squares using a scalpel. These were then affixed 12 mm from the left hand
edge of a plain glass slide (VWR International, UK) using UHU glue (UHU
GmbH, Germany), and allowed to air dry for 10 minutes. A metal grid was
used to hold 12 slides in predefined and consistent positions on the
application plate of a Camag Automatic TLC Sampler 4 (Camag,
Switzerland). The winCATS planer chromatography management software
(Camag, Switzerland) was used to write programs which result in the
application of duplicate spots of 0.1 μl of 100 μl/ml ganglioside
or ganglioside complex over a predefined 0.4 μm2 area. An example
of a 10×10 grid is shown in FIG. 1A. Larger grids of 23×23
spots have also been produced, spread over 2 separate slides. Printed
hydrophobic supports were outlined with a hydrophobic barrier pen (Vector
Laboratories, UK) and allowed to air dry for 20 minutes. They were then
stored overnight at 4° C. before use.

[0150] Membranes were blocked in at least 100 ml/cm2 of 2% bovine
serum albumin/phosphate buffer saline (BSA/PBS) for 1 hr at 4° C.
Serum samples, CSF, monoclonal antibodies, siglec-Fc fusion proteins
(preconjugated to horse radish peroxidase (HRP) linked anti-Fc antibody),
or HRP-bacterial toxin conjugates were diluted in 1%; BSA/PBS. 250 μl
of this diluted sample was then applied to a pre-printed membrane and
incubated at 4° C. After 1 hr, the sample was tipped from the
membrane and the slides were briefly placed back in the 2% BSA/PBS
blocking solution. Probes requiring a secondary antibody underwent a
primary wash phase. These membranes were transferred to at least 500
ml/cm2 of 1% BSA/PBS for 15 minutes of washing on a shaker set at
100 rpm. This process was repeated once. These membranes were tapped dry,
250 μl of the appropriate HRP linked secondary antibody was applied
(diluted in 1% BSA/PBS), and incubated for 30 m at 4° C. All
membranes then entered a wash phase. For probes not requiring a secondary
antibody (siglecs and HRP-conjugated bacterial toxins), this immediately
followed the primary incubation.

[0151] This wash phase consisted of two changes of 1% BSA and three
changes of PBS, again each of at least 500 ml/cm2. BSA washes were
of 30 m duration, PBS for 5 m, both on a shaker set at 100 rpm. Slides
were then briefly dipped in two changes of distilled water (500
ml/cm2). A chemiluminescent detection reaction was then performed
using ECL plus (Amersham/GE Healthcare, UK), made up according to
manufacturer's instructions. 450 μl of this detection solution was
then applied to the membranes and left for 3 minutes at room temperature.
The solution was tipped from the membranes and signal was detected on
radiographic film. Exposure time was initially 15 s; subsequent exposures
were adjusted on the basis of this first result. Films were digitised by
flatbed scanning and the images analysed and quantified by the array
analysis component of ImageQuant TL software (Amersham Biosciences, UK).
Examples of processed membranes are shown in FIGS. 2 and 3, as described
in detail elsewhere in Examples 2 and 3 below. An example of array
analysis is shown in FIG. 4 and described elsewhere.

Example 1

Combinatorial GSL/Lipid Grids

[0152] Examples of combinatorial GSL/lipid grids are shown in FIGS. 1A and
1B, which show 10×10 and 23×23 grids, respectively. A line of
methanol as negative control runs diagonally across the membrane from top
left to bottom right corners. This acts as a line of symmetry for
duplicate spots within the membrane. The first row and first column
contain single species. Other spots are complexes of two GSLs, and
consist of the single glycolipid spotted at the extreme left of the row
combined with the glycolipid at the top of the column.

Example 2

Processed Combinatorial Lipid Grids

[0153] A 10×10 GSL combinatorial grid was probed with serum from a
patient with an inflammatory neuropathy as the primary probe, followed by
an anti-human IgG-HRP linked secondary antibody and then a development
step. The prominent positive spot corresponds to the ganglioside complex
GM1/GQ1b (see FIG. 2A).

[0154] FIG. 2B shows a 23×23 combinatorial lipid grid in which serum
from a patient with an undiagnosed neurological disorder was used as the
primary probe, followed by with anti-human IgG-HRP linked secondary
antibody and then a development step. Sulphatide is spotted in Row 2 and
Column 2 and is bound by IgG antibody in this serum when on its own (spot
1,2 and spot 2,1) and when in combination with other lipids (e.g. Spot
2,3; 2,4; 2,19 and 2, 22; in corresponding rows and columns). It should
be noted that the combination of sulphatide when complexed with
glycolipids spotted at positions 5 through 17 creates an inhibitory
interaction that prevents the anti-sulphatide IgG from binding
sulphatide. The circled spots show binding to complexes of lipid pair
comprising digalactosyl diglyceride/cholesterol and phosphatidyl
inositol/cholesterol. Note that neither of these 3 lipids is bound when
spotted on its own (i.e. 1,4; 1,18; and 1,21 positions in corresponding
rows and columns are negative). The prominent black signal circa position
6,12 is a technical artefact.

[0155] FIG. 2C shows a 23×23 combinatorial lipid grid probed with
serum from a patient with multiple sclerosis. The prominent positive spot
corresponds to the complex of phosphatidyl inositol/cholesterol and is
symmetrically present at 4,21. The serum does not react with either
phosphatidyl inositol (spot or cholesterol alone (i.e. positions 1,21 and
1,4 are negative). The black signals circa positions 3,4; 4,15 and 5,5
are technical artefacts.

Example 3

Alternative Patterns of Ganglioside Binding

[0156] In FIG. 3A, a complicated pattern of binding is demonstrated for
siglec-E. Some ganglioside pairings attenuate signals obtained with
either ganglioside alone and some enhance the signal (e.g. the GM3 signal
is attenuated by GM1 and enhanced by GD1a). Intensity data is plotted in
FIGS. 4A and 4B. In FIG. 3B, the monoclonal mouse anti-GQ1b antibody mAb
MOG26 is shown to bind GQ1b on its own and in the presence of other GSLs.
It also binds a combination of GD1b and GM3 whilst binding negligibly to
either ganglioside alone. In FIG. 3C, cholera toxin is shown to bind well
to GM1 and GT1a, either alone or in combination with other GSLs. GD1a
creates a relatively inhibitory environment for cholera toxin binding,
suppressing the binding intensity with both GM1 and GT1a.

Example 4

Array Analysis

[0157] Processed array grids are analysed using ImageQuant software
(Amersham Biosciences), which produces a large amount of intensity data
(see FIG. 4A). This, along with pictorial representation, is used to
identify ganglioside pairs of interest for further evaluation (see FIG.
4B).

[0158] Alternatively, binding to a particular ganglioside series of
complexes can be compared for different binding agents. For example,
binding of anti-GM1 mAb DG1 is inhibited in the presence of any other
paired species, whereas cholera toxin is able to bind regardless of the
presence or absence of a second ganglioside (see FIG. 4C).

[0159] Anti-GM1 mAbs DG1 and DG2 were applied to PVDF membranes or to
ELISA plates at a concentration of 1 mg/ml. Using ELISA, DG1 (left panel)
binds to GM1 alone, but with a weak signal for GM1/GD1a complex. DG2
(right panel) binds GM1 and is much less inhibited by the presence of
GD1a (see FIG. 5A). Quantitative ELISA results from 4 independent
experiments are shown in FIG. 5C. When using PVDF-glycoarrays, DG1 did
not bind to complexes of GM1/GD1a, but bound to GM1 alone (see left hand
panel of FIG. 5B), whereas DG2 bound to complexes of GM1/GD1a, as well as
to GM1 alone (see right hand panel of FIG. 5B). Therefore, as can be seen
in FIG. 5D, the inhibitory effect of GM1/GSL complexes on antibody
binding is greater for DG1 than for DG2. These experiments show that the
difference in the behaviour of the two antibodies with respect to binding
of GM1 and GM1/GD1a complexes is more marked on PVDF as compared to
ELISA.

[0160] Consistent with the results from the PVDF-based assays, DG1 failed
to bind to nerve terminals in living tissue in which GM1/GSL complexes
are thought to form (see FIG. 6G). The nerve terminals were identified by
staining with bungarotoxin and detection using the cholera toxin was used
to confirm expression of GM1 at these nerve terminals (see FIGS. 6 B, E,
H and C). In contrast, DG2 was able to bind GM1 in the nerve terminals of
living tissue (see FIG. 6D), confirming that the detection method is
working in vivo. Thus, the PVDF-based method is able to identify that the
DG1 antibody (which does not bind to GM1 in living tissue) is unable to
bind to GM1 complexes, whereas ELISA is not able to discriminate as
effectively. Therefore, the PVDF-based assay may be more representative
of the in vivo situation, where GM1 is thought to exist as a complex,
than the ELISA-based assay.

Example 6

A Comparison of mAb MOG26 and Siglec-E Reactivities on ELISA and
PVDF-Glycoarrays

[0161] Identical preparations of MOG26 (FIGS. 7A and B) and siglec-E-Fc
(FIGS. 7C and D) were investigated by ELISA and PVDF arrays. On ELISA,
MOG26 reacts strongly with GM1/GD1a (see FIG. 7B), yet no signal is seen
on PVDF (see FIG. 7A) for this complex (enclosed by circles) even when
the antibody concentration is doubled form that used on ELISA and the
exposure time is increased to 5 minutes. This reflects the situation seen
in the live membrane of the GD3s.sup.-/- mouse (in which GM1/GD1a
complexes are expected to form) where this mAb also fails to bind (data
not shown). Siglec-E binds GT1b/GM2 complex (enclosed by circles) in the
PVDF system (see FIG. 7C), as well as showing reactivity towards GM2 (and
to a lesser extent GT1b). On ELISA (see FIG. 7D), the signal is barely
above baseline for GM2/GT1b, and absolutely undetectable for GM2 and GT1b
alone. The graph is plotted on the same scale as for MOG26. These
experiments demonstrate that reactivities can be seen on PVDF which are
not replicated on ELISA, and vice versa.